In recent years, a myriad of reports have appeared on the attempted specific targeting of tumor cells with monoclonal antibody-drug conjugates (R. V. J. Chari., 31 Adv. Drug Deliv. Res., 89-104 (1998); G. A. Pietersz and K. Krauer, 2 J. Drug Targeting 183-215 (1994); Sela et al., in Immunoconjugates 189-216 (C. Vogel, ed. 1987); Ghose et al., in Targeted Drugs 1-22 (E. Goldberg, ed. 1983); Diener et al., in Antibody mediated delivery systems 1-23 (J. Rodwell, ed. 1988); Pietersz et al., in Antibody mediated delivery systems 25-53 (J. Rodwell, ed. 1988); Bumol et al., in Antibody mediated delivery system 55-79 (J. Rodwell, ed. 1988). Cytotoxic drugs such as methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, and chlorambucil have been conjugated to a variety of murine monoclonal antibodies. In some cases, the drug molecules were linked to the antibody molecules through an intermediary carrier molecule such as serum albumin (Garnett et al., 46 Cancer Res. 2407-2412 (1986); Ohkawa et al., 23 Cancer Immumol. Immunother. 81-86 (1986); Endo et al., 47 Cancer Res. 1076-1080 (1980)), dextran (Hurwitz et al., 2 Appl. Biochem. 25-35 (1980); Manabi et al., 34 Biochem. Pharmacol. 289-291 (1985); Dillman et al., 46 Cancer Res. 4886-4891 (1986); Shoval et al., 85 Proc. Natl. Acad. Sci. 8276-8280 (1988)), or polyglutamic acid (Tsukada et al., 73 J. Natl. Canc. Inst. 721-729 (1984); Kato et al., 27 J. Med. Chem. 1602-1607 (1984); Tsukada et al., 52 Br. J. Cancer 111-116 (1985)).
A wide array of linker technologies have been employed for the preparation of such immunoconjugates and both cleavable and non-cleavable linkers have been investigated. In most cases, the full cytotoxic potential of the drugs could only be observed, however, if the drug molecules could be released from the conjugates in unmodified form at the target site.
One of the cleavable linkers that has been employed for the preparation of antibody-drug conjugates is an acid-labile linker based on cis-aconitic acid that takes advantage of the acidic environment of different intracellular compartments such as the endosomes encountered during receptor mediated endocytosis and the lysosomes. Shen and Ryser introduced this method for the preparation of conjugates of daunorubicin with macromolecular carriers (102 Biochem. Biophys. Res. Commun. 1048-1054 (1981)). Yang and Reisfeld used the same technique to conjugate daunorubicin to an anti-melanoma antibody (80 J. Natl. Canc. Inst. 1154-1159 (1988)). Dillman et al. also used an acid-labile linker in a similar fashion to prepare conjugates of daunorubicin with an anti-T cell antibody (48 Cancer Res. 6097-6102 (1988)). Trail et al. linked doxorubicin to antibodies via an acid-labile hydrazone bond (52 Cancer Res. 5693-5700 (1992)).
An alternative approach, explored by Trouet et al., involved linking daunorubicin to an antibody via a peptide spacer arm (79 Proc. Natl. Acad. Sci. 626-629 (1982)). This was done under the premise that free drug could be released from such a conjugate by the action of lysosomal peptidases.
In vitro cytotoxicity tests, however, have revealed that antibody-drug conjugates rarely achieved the same cytotoxic potency as the free unconjugated drugs. This suggested that mechanisms by which drug molecules are released from the antibodies are very inefficient. In the area of immunotoxins, conjugates formed via disulfide bridges between monoclonal antibodies and catalytically active protein toxins were shown to be more cytotoxic than conjugates containing other linkers. See, Lambert et al., 260 J. Biol. Chem. 12035-12041 (1985); Lambert et al., in Immunotoxins 175-209 (A. Frankel, ed. 1988); Ghetie et al., 48 Cancer Res. 2610-2617 (1988). This was attributed to the high intracellular concentration of glutathione contributing to the efficient cleavage of the disulfide bond between an antibody molecule and a toxin. Despite this, there are only a few reported examples of the use of disulfide bridges for the preparation of conjugates between drugs and macromolecules. Shen et al. described the conversion of methotrexate into a mercaptoethylamide derivative followed by conjugation with poly-D-lysine via a disulfide bond (260 J. Biol. Chem. 10905-10908 (1985)). A recent report described the preparation of a conjugate of the trisulfide-containing toxic drug calicheamicin with an antibody (L. M. Hinman et al., 53 Cancer Res. 3336-3342 (1993); E. L. Sievers et al., 93 Blood 3678-3684 (1999).
One reason for the lack of disulfide linked antibody-drug conjugates is the unavailability of cytotoxic drugs possessing a sulfur atom containing moiety that can be readily used to link the drug to an antibody via a disulfide bridge. Furthermore, chemical modification of existing drugs is difficult without diminishing their cytotoxic potential.
Another major drawback with existing antibody-drug conjugates is their inability to deliver a sufficient concentration of drug to the target site because of the limited number of targeted antigens and the relatively moderate cytotoxicity of cancerostatic drugs like methotrexate, daunorubicin and vincristine. For example, an antibody conjugate of doxorubicin was evaluated in human clinical trials, and found to be ineffective (Tolcher et al., 17 J. Clinical Oncol. 478-484 (1999)). In order to achieve significant cytotoxicity, linkage of a large number of drug molecules either directly to the antibody or through a polymeric carrier molecule becomes necessary. However, such heavily modified antibodies often display impaired binding to the target antigen and fast in vivo clearance from the blood stream.
Maytansinoids are highly cytotoxic drugs. Maytansine was first isolated by Kupchan et al. from the east African shrub Maytenus serrata and shown to be 100 to 1000 fold more cytotoxic than conventional cancer chemotherapeutic agents like methotrexate, daunorubicin, and vincristine (U.S. Pat. No. 3,896,111). Subsequently it was discovered that some microbes also produce maytansinoids, such as maytansinol and C-3 esters of maytansinol (U.S. Pat. No. 4,151,042). Synthetic C-3 esters of maytansinol and analogues of maytansinol have also been reported (Kupchan et al., 21 J. Med. Chem. 31-37 (1978); Higashide et al., 270 Nature 721-722 (1977); Kawai et al., 32 Chem. Pharm. Bull. 3441-3451 (1984)). Examples of analogues of maytansinol from which C-3 esters have been prepared include maytansinol with modifications on the aromatic ring (e.g. dechloro) or at the C-9, C-14 (e.g. hydroxylated methyl group), C-15, C-18, C-20 and C-4,5.
The naturally occurring and synthetic C-3 esters can be classified into two groups:
(a) C-3 esters with simple carboxylic acids (U.S. Pat. Nos. 4,248,870; 4,265,814; 4,308,268; 4,308,269; 4,309,428; 4,317,821; 4,322,348; and 4,331,598), and PA1 (b) C-3 esters with derivatives of N-methyl-L-alanine (U.S. Pat. Nos. 4,137,230; 4,260,608; 5,208,020; 5,416,064; and 12 Chem. Pharm. Bull. 3441 (1984)). PA1 (1) conducting reductive hydrolysis of a maytansinoid C-3 ester with a reducing agent selected from the group consisting of lithium trimethoxyaluminum hydride (LiAl(OMe).sub.3 H), lithium triethoxyaluminum hydride (LiAl(OEt).sub.3 H), lithium tripropoxyaluminum hydride (LiAl(OPr).sub.3 H), sodium trimethoxyaluminum hydride (NaAl(OMe).sub.3 H), sodium triethoxyaluminum hydride (NaAl(OEt).sub.3 H) and sodium tripropoxyaluminum hydride (NaAl(OPr).sub.3 H), to yield a maytansinol; PA1 (2) purifying the maytansinol to remove side products when present; PA1 (3) esterifying the purified maytansinol with a carboxylic acid to yield a reaction mixture of an L- and a D-aminoacyl ester of maytansinol; PA1 (4) separating the L-aminoacyl ester of maytansinol from the reaction mixture in (3); PA1 (5) reducing the L-aminoacyl ester of maytansinol to yield a thiol-containing maytansinoid; and PA1 (6) purifying the thiol-containing maytansinoid.
Esters of group (b) were found to be much more cytotoxic than esters of group (a).
Maytansine is a mitotic inhibitor. Treatment of L1210 cells in vivo with maytansine has been reported to result in 67% of the cells accumulating in mitosis. Untreated control cells were reported to demonstrate a mitotic index ranging from between 3.2 to 5.8% (Sieber et al., 43 Comparative Leukemia Research 1975, Bibl. Haemat. 495-500 (1976)). Experiments with sea urchin eggs and clam eggs have suggested that maytansine inhibits mitosis by interfering with the formation of microtubules through the inhibition of the polymerization of the microtubule protein, tubulin (Remillard et al., 189 Science 1002-1005 (1975)).
In vitro P388, L1210, and LY5178 murine leukemic cell suspensions have been found to be inhibited by maytansine at doses of 10.sup.-3 to 10.sup.-1 microgram/mL, with the P388 line being the most sensitive. Maytansine has also been shown to be an active inhibitor of in vitro growth of human nasopharyngeal carcinoma cells. The human acute lymphoblastic leukemia line C.E.M. was reported inhibited by concentrations as low as 10.sup.-7 microgram/ml (Wolpert-DeFillippes et al., 24 Biochem. Pharmacol. 1735-1738 (1975)).
In vivo, maytansine has also been shown to be active. Tumor growth in the P388 lymphocytic leukemia system was shown to be inhibited over a 50- to 100-fold dosage range which suggested a high therapeutic index; also significant inhibitory activity could be demonstrated with the L1210 mouse leukemia system, the human Lewis lung carcinoma system and the human B-16 melanocarcinoma system (Kupchan, 33 Ped. Proc. 2288-2295 (1974)).
Because the maytansinoids are highly cytotoxic, they were expected to be of use in the treatment of many diseases such as cancer. This expectation has yet to be realized. Clinical trials with maytansine were not favorable due to a number of side effects (Issel et al., 5 Can. Trtmnt. Rev. 199-207 (1978)). Adverse effects to the central nervous system and gastrointestinal symptoms were responsible for some patients refusing further therapy (Issel at 204), and it appeared that maytansine was associated with peripheral neuropathy that might be cumulative (Issel at 207).
However, forms of maytansinoids that are highly cytotoxic, yet can still effectively be used in the treatment of many disease, have been described (U.S. Pat. Nos. 5,208,020 and 5,416,064; Chari et al., 52 Cancer Res. 127-131 (1992); Liu et al., 93 Proc. Natl. Acad. Sci. 8618-8623 (1996)).
A further drawback to the therapeutic use of maytansinoids, of interest here, is that the process for the preparation and purification of thiol-containing maytansinoids involves several inefficient chromatographic steps that are cumbersome, not easily scalable and result in only moderate yields.
U.S. Pat. Nos. 5,208,020 and 5,416,064 disclose that a thiol-containing maytansinoid may be produced by first converting a maytansinoid bearing an ester group into maytansinol, then esterifying the resulting maytansinol with N-methyl-L-alanine or N-methyl-L-cysteine derivatives to yield disulfide-containing maytansinoids, followed by cleavage of the disulfide group with dithiothreitol to the thiol-containing maytansinoids. However, this process involves several inefficient steps that are cumbersome and result in moderate yields.
More specifically, maytansinol is first derived from maytansine or other esters of maytansinol by reduction, such as with lithium aluminum hydride. (Kupchan, S. M. et al., 21 J. Med. Chem. 31-37 (1978); U.S. Pat. No. 4,360,462). It is also possible to isolate maytansinol from the microorganism Nocardia (see Higashide et al., U.S. Pat. No. 4,151,042). In a specific example, the conversion of Ansamitocin P-3 into maytansinol by reductive hydrolysis with lithium aluminum hydride in tetrahydrofuran at -5.degree. C. was described in U.S. Pat. No. 4,162,940. However, the reaction with lithium aluminum hydride results in the formation of several side products which can be removed only by careful preparative thin layer chromatography on silica gel. Thus, this process is not amenable for industrial scale use.
The next step in the process is the conversion of maytansinol to different ester derivatives using N-methyl-L-alanine or N-methyl-L-cysteine derivatives, and suitable agents such as dicyclohexylcarbodiimide (DCC) and catalytic amounts of zinc chloride (see U.S. Pat. No. 4,137,230; Kawai et al., 32 Chem. Pharm. Bull. 3441-3951 (1984); U.S. Pat. No. 4,260,609). Two diastereomeric products containing the D and L-aminoacyl side chains result, as does a small portion of unreacted maytansinol. While the unreacted maytansinol is readily separated from its esters by column chromatography on silica gel, the diastereomeric maytansinoid esters are barely separable. In the process previously described (Kupchan, S. M., 21 J. Med. Chem. 31-37 (1978); U.S. Pat. No. 4,360,462), the desired L-aminoacyl ester is obtained after purification over two silica gel columns followed by further purification by preparative thin layer chromatography on silica gel. Thus, this process is also not amenable for industrial scale use.
The last step in the process, the reduction of the disulfide-containing maytansinoids to the corresponding thiol-containing maytansinoids, is achieved by treatment with dithiotlireitol (DTT), purification by HPLC using a C-18 column and elution with a linear gradient of 55% to 80% acetonitrile in H.sub.2 O. However, this step results in low yields and is also not amenable for industrial scale use. Thiol-containing maytansinoids are not very soluble in the ethanol/water solvent mixture used for the reaction. Furthermore, purification by HPLC on a reverse phase C-18 column using acetonitrile/water as the mobile phase results in poor recovery and can result in the dimerization of some product.
Accordingly, an improved process for the preparation and purification of thiol-containing maytansinoids, that reduces the complexity of the process, allows scalability and increases the yield, is greatly needed.